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Northern Blotting: Presented TO
Northern Blotting: Presented TO
Presented TO:
Dr. Dildar Hussain Kalhoro
Associate Professor
Department OF Veterinary Microbiology
Only mRNA from the cell types that are synthesizing the protein will
hybridize to the probe.
Procedure
STEP 1 : ISOLATAION OF RNA :
All the target RNA’s molecules should be extracted from the
sample.
Isolate RNA from cells or tissue samples using the TRI
reagent or genelute™ kit for mammalian cells or tissues.
Agarose & Buffer : Agarose & buffer are used is a gel to conduct
electrical current.
Formaldehyde :
Formaldehyde is used to unfragment the branched RNA molecule to
simple linear one and to prevent it form coiling again.
Gel Electrophoresis
Blotting/ Transfer
The transfer or blotting is the step in
which the mRNA from the
electrophoresis gel will be transferred
onto a nylon membrane so it may be
accessible to a probe for hybridization
and detection. The separated mRNA
bands are then blotted on chemically
reactive filter paper.
Blotting/ Transfer
Traditionally, a nitrocellulose membrane is used, although
nylon or a positively charged nylon membrane may be used.